Correlated production and consumption of chloromethane in the Arabidopsis thaliana phyllosphere

Chloromethane (CH3Cl) is a toxic gas mainly produced naturally, in particular by plants, and its emissions contribute to ozone destruction in the stratosphere. Conversely, CH3Cl can be degraded and used as the sole carbon and energy source by specialised methylotrophic bacteria, isolated from a variety of environments including the phyllosphere, i.e. the aerial parts of vegetation. The potential role of phyllospheric CH3Cl-degrading bacteria as a filter for plant emissions of CH3Cl was investigated using variants of Arabidopsis thaliana with low, wild-type and high expression of HOL1 methyltransferase previously shown to be responsible for most of CH3Cl emissions by A. thaliana. Presence and expression of the bacterial chloromethane dehalogenase cmuA gene in the A. thaliana phyllosphere correlated with HOL1 genotype, as shown by qPCR and RT-qPCR. Production of CH3Cl by A. thaliana paralleled HOL1 expression, as assessed by a fluorescence-based bioreporter. The relation between plant production of CH3Cl and relative abundance of CH3Cl-degrading bacteria in the phyllosphere suggests that CH3Cl-degrading bacteria co-determine the extent of plant emissions of CH3Cl to the atmosphere

Hydrogen and carbon isotope fractionation during degradation of chloromethane by methylotrophic bacteria

Chloromethane (CH3Cl) is a widely studied volatile halocarbon involved in the destruction of ozone in the stratosphere. Nevertheless, its global budget still remains debated. Stable isotope analysis is a powerful tool to constrain fluxes of chloromethane between various environmental compartments which involve a multiplicity of sources and sinks, and both biotic and abiotic processes. In this study, we measured hydrogen and carbon isotope fractionation of the remaining untransformed chloromethane following its degradation by methylotrophic bacterial strains Methylobacterium extorquens CM4 and Hyphomicrobium sp. MC1, which belong to different genera but both use the cmu pathway, the only pathway for bacterial degradation of chloromethane characterized so far. Hydrogen isotope fractionation for degradation of chloromethane was determined for the first time, and yielded enrichment factors (epsilon) of -29 parts per thousand and -27 parts per thousand for strains CM4 and MC1, respectively. In agreement with previous studies, enrichment in C-13 of untransformed CH3Cl was also observed, and similar isotope enrichment factors (e) of -41 parts per thousand and -38 parts per thousand were obtained for degradation of chloromethane by strains CM4 and MC1, respectively. These combined hydrogen and carbon isotopic data for bacterial degradation of chloromethane will contribute to refine models of the global atmospheric budget of chloromethane

Tetrachloromethane-degrading bacterial enrichment cultures and isolates from a contaminated aquifer

The prokaryotic community of a groundwater aquifer exposed to high concentrations of tetrachloromethane (CCl₄) for more than three decades was followed by terminal restriction fragment length polymorphism (T-RFLP) during pump-and-treat remediation at the contamination source. Bacterial enrichments and isolates were obtained under selective anoxic conditions, and degraded 10 mg·L(-1) CCl₄, with less than 10% transient formation of chloroform. Dichloromethane and chloromethane were not detected. Several tetrachloromethane-degrading strains were isolated from these enrichments, including bacteria from the Klebsiella and Clostridium genera closely related to previously described CCl₄ degrading bacteria, and strain TM1, assigned to the genus Pelosinus, for which this property was not yet described. Pelosinus sp. TM1, an oxygen-tolerant, Gram-positive bacterium with strictly anaerobic metabolism, excreted a thermostable metabolite into the culture medium that allowed extracellular CCl₄ transformation. As estimated by T-RFLP, phylotypes of CCl₄-degrading enrichment cultures represented less than 7%, and archaeal and Pelosinus strains less than 0.5% of the total prokaryotic groundwater community

Methylobacterium Genome Sequences: A Reference Blueprint to Investigate Microbial Metabolism of C1 Compounds from Natural and Industrial Sources

Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared. Methodology/Principal Findings The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name “island integration determinant” (iid).Conclusion/Significance These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.Organismic and Evolutionary Biolog

The 380 kb pCMU01 Plasmid Encodes Chloromethane Utilization Genes and Redundant Genes for Vitamin B₁₂- and Tetrahydrofolate-Dependent Chloromethane Metabolism in <i>Methylobacterium extorquens</i> CM4: A Proteomic and Bioinformatics Study

<div><p>Chloromethane (CH₃Cl) is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for bacterial chloromethane utilization (<i>cmu</i>) was characterized in <i>Methylobacterium extorquens</i> CM4, a methylotrophic bacterium able to utilize compounds without carbon-carbon bonds such as methanol and chloromethane as the sole carbon source for growth. Previous work demonstrated that tetrahydrofolate and vitamin B₁₂ are essential cofactors of <i>cmuA</i>- and <i>cmuB</i>-encoded methyltransferases of chloromethane dehalogenase, and that the pathway for chloromethane utilization is distinct from that for methanol. This work reports genomic and proteomic data demonstrating that cognate <i>cmu</i> genes are located on the 380 kb pCMU01 plasmid, which drives the previously defined pathway for tetrahydrofolate-mediated chloromethane dehalogenation. Comparison of complete genome sequences of strain CM4 and that of four other <i>M. extorquens</i> strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (<i>bluB2</i>, <i>btuB</i>, <i>cobA</i>, <i>cbiD</i>), as well as 13 duplicated genes with homologs of chromosome-borne genes involved in vitamin B₁₂-associated biosynthesis and transport, or in tetrahydrofolate-dependent metabolism (<i>folC2</i>). In addition, the presence of both chromosomal and plasmid-borne genes for corrinoid salvaging pathways may ensure corrinoid coenzyme supply in challenging environments. Proteomes of <i>M. extorquens</i> CM4 grown with one-carbon substrates chloromethane and methanol were compared. Of the 49 proteins with differential abundance identified, only five (CmuA, CmuB, PurU, CobH2 and a PaaE-like uncharacterized putative oxidoreductase) are encoded by the pCMU01 plasmid. The mainly chromosome-encoded response to chloromethane involves gene clusters associated with oxidative stress, production of reducing equivalents (PntAA, Nuo complex), conversion of tetrahydrofolate-bound one-carbon units, and central metabolism. The mosaic organization of plasmid pCMU01 and the clustering of genes coding for dehalogenase enzymes and for biosynthesis of associated cofactors suggests a history of gene acquisition related to chloromethane utilization.</p></div

Fluorescence-Based Bacterial Bioreporter for Specific Detection of Methyl Halide Emissions in the Environment

Methyl halides are volatile one-carbon compounds responsible for substantial depletion of stratospheric ozone. Among them, chloromethane (CH3Cl) is the most abundant halogenated hydrocarbon in the atmosphere. Global budgets of methyl halides in the environment are still poorly understood due to uncertainties in their natural sources, mainly from vegetation, and their sinks, which include chloromethane-degrading bacteria. A bacterial bioreporter for the detection of methyl halides was developed on the basis of detailed knowledge of the physiology and genetics of Methylobacterium extorquens CM4, an aerobic alphaproteobacterium which utilizes chloromethane as the sole source of carbon and energy. A plasmid construct with the promoter region of the chloromethane dehalogenase gene cmuA fused to a promotorless yellow fluorescent protein gene cassette resulted in specific methyl halide-dependent fluorescence when introduced into M. extorquens CM4. The bacterial whole-cell bioreporter allowed detection of methyl halides at femtomolar levels and quantification at concentrations above 10 pM (approximately 240 ppt). As shown for the model chloromethane-producing plant Arabidopsis thaliana in particular, the bioreporter may provide an attractive alternative to analytical chemical methods to screen for natural sources of methyl halide emissions

Tetrachloromethane-Degrading Bacterial Enrichment Cultures and Isolates from a Contaminated Aquifer

The prokaryotic community of a groundwater aquifer exposed to high concentrations of tetrachloromethane (CCl4) for more than three decades was followed by terminal restriction fragment length polymorphism (T-RFLP) during pump-and-treat remediation at the contamination source. Bacterial enrichments and isolates were obtained under selective anoxic conditions, and degraded 10 mg·L−1 CCl4, with less than 10% transient formation of chloroform. Dichloromethane and chloromethane were not detected. Several tetrachloromethane-degrading strains were isolated from these enrichments, including bacteria from the Klebsiella and Clostridium genera closely related to previously described CCl4 degrading bacteria, and strain TM1, assigned to the genus Pelosinus, for which this property was not yet described. Pelosinus sp. TM1, an oxygen-tolerant, Gram-positive bacterium with strictly anaerobic metabolism, excreted a thermostable metabolite into the culture medium that allowed extracellular CCl4 transformation. As estimated by T-RFLP, phylotypes of CCl4-degrading enrichment cultures represented less than 7%, and archaeal and Pelosinus strains less than 0.5% of the total prokaryotic groundwater community

Gene redundancy for cobalamin and tetrahydrofolate metabolism in <i>M. extorquens</i> CM4.

a<p>Homologs with >90% aa Id (with mentioned exceptions) found in the chromosome of all <i>M. extorquens</i> strains AM1, BJ001, DM4, and PA1 (common core genome), in one of the strains (shared accessory genome), or none of these strains (CM4 specific CDS). The accessory genome includes a <i>btuB</i> homolog (Mpop_3807, 65% aa Id) in strain BJ001. For strain AM1, a putative dihydrofolate reductase <i>dfrB</i> gene (META2_0242, 34 and 28% aa Id with DmrA and DfrA, respectively) is found in addition to the chromosomal gene; moreover, homologs to Mchl_1923 (META2_0462, 33% aa Id with the N-terminal domain), and CzcA2 (META2_1026, 85% aa Id with pCMU01 plasmid <i>czcA2</i>) are found.</p>b<p>MaGe annotation (<a href="https://www.genoscope.cns.fr/agc/microscope" target="_blank">https://www.genoscope.cns.fr/agc/microscope</a>).</p>c<p>Precursors are uroporphyrinogen III and 5,6-dimethylbenzimidazole.</p>d<p>n.d., not detected.</p>e<p>Encode for homologs of different length: CobA (267 aa)/CysG (485 aa); CobC2 (519 aa)/CobC (338 aa); PurU (287 aa)/PurN (219 aa).</p>f<p>In <i>M. extorquens</i> strains, H₄F is synthesized either <i>de novo</i> or salvaged from 5,10-methenyl-H₄F, or 5- or 10-formyl-H₄F <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Vuilleumier2" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Maden1" target="_blank">[72]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Vorholt1" target="_blank">[73]</a>.</p

Methylotrophic metabolism and chloromethane utilization pathway in <b><i>Methylobacterium extorquens</i></b><b> CM4.</b>

<p>The left-hand scale indicates carbon oxidation state. The <u>c</u>hloro<u>m</u>ethane <u>u</u>tilization <i>cmu</i> pathway (bold arrows) funnels the chloromethane-derived methyl group into central metabolism via methylene-H₄F (CH₂ = H₄F), while the methanol (CH₃OH) oxidation pathway operates with formaldehyde (HCHO) as a metabolic intermediate (grey arrows). H₄F- and H₄MPT-dependent enzyme-mediated steps are depicted in blue and pink, respectively. Carbon assimilation operates via the serine cycle (Ser) coupled with the ethylmalonyl-CoA pathway (EMCP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Peyraud2" target="_blank">[67]</a>. Spontaneous condensation of HCHO with H₄F or H₄MPT, and formaldehyde oxidation to methylene-H₄F are shown with broken line. In the <i>cmu</i> pathway, the methyl group enters a specific H₄F-oxidation pathway for energy production driven by the FolD and PurU enzymes. Protein-encoded genes or genes located on plasmid pCMU01 are shown in bold. Boxes and circles highlight proteins more abundant in chloromethane- and methanol grown-cultures, respectively. CmuA, methyltransferase/corrinoid-binding two-domain protein; CmuB, methylcobalamin:H₄F methyltransferase; Fae, formaldehyde activating enzyme; Fch, methenyl-H₄F cyclohydrolase; FDHs, formate dehydrogenases; Fhc, formyltransferase-hydrolase complex; FolD, bifunctional methylene-H₄F dehydrogenase/cyclohydrolase; FtfL, formate-H₄F ligase; Gck, glycerate kinase; GcvT, H₄F-dependent aminomethyltransferase; HprA, hydroxypyruvate reductase; MDH, methanol dehydrogenase; MetF, methylene-H₄F reductase; MtdA, bifunctional NAD(P)-dependant methylene-H₄F and methylene-H₄MPT dehydrogenase; MtdB, NAD(P)-dependent methylene-H₄MPT dehydrogenase; Mch, methenyl-H₄MPT cyclohydrolase; MtkA, malate thiokinase large subunit; MxaF, MDH alpha subunit, PurU, 10-formyl-H₄F hydrolase; Sga, serine-glyoxylate aminotransferase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Chistoserdova1" target="_blank">[12]</a>. Plasmid pCMU01 encoded proteins with predicted functions include putative uncharacterized methyltransferases CmuC and CmuC2, the putative PaaE-like oxidoreductase, and the putative PQQ-linked dehydrogenase of unknown specificity XoxF2. GvcT may serve to transfer methyl groups from a wide range of substrates to H₄F, as proposed for members that belong to the COG0354-related enzymes such as YgfZ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Halsey1" target="_blank">[68]</a>.</p

Analysis of the theoretical proteome of plasmid pCMU01.

a<p>Compared predicted proteome sizes are, <i>M. extorquens</i> strains AM1, 6531 proteins (genome sequence accession no NC_012808); DM4, 5773 proteins (NC_012988); PA1, 5357 proteins (NC_01017); CM4, 6454 proteins (NC_011757); BJ001, 6027 proteins (NC_010725). Homologous proteins were defined as proteins with at least 40% identity covering over 80% of the sequence. Three classes of proteins were considered: Unique, 157 pCMU01 plasmid-encoded proteins without homologs in any of the compared genomes, including the chromosome and the second plasmid p2MCHL of strain CM4; Common, 56 pCMU01 plasmid-encoded proteins with homologs on the chromosome of all 5 <i>M. extorquens</i> genomes including that of strain CM4; Occasional, 173 pCMU01 plasmid-encoded proteins with homologs in at least one of the 5 <i>M. extorquens</i> genomes. Plasmid pCMU01 and plasmid p1METDI of strain DM4 share 56 homologs localized on three gene clusters. Selected examples are indicated when relevant.</p>b<p>CmuC/CmuC2 homologs share less homologies between them (31% aa Id) than with homologs found in other chloromethane-degrading <i>Hyphomicrobium</i> strains: 40% with strain CM2 CmuC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Borodina1" target="_blank">[71]</a> and 37% aa Id with strain MC1 CmuC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Vuilleumier1" target="_blank">[4]</a>. <i>M. extorquens</i> CM4 is the only chloromethane-degrading strain so far which contains two methyltransferase-encoding <i>cmuC</i> genes of unknown function. Transposon insertion in gene <i>cmuC</i> was previously demonstrated to prevent strain CM4 growth with chloromethane <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Vannelli2" target="_blank">[10]</a>.</p>c<p>pCMU01 plasmid encoded protein MetF2 (Mchl_5726) previously demonstrated to be essential for chloromethane utilization <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056598#pone.0056598-Studer1" target="_blank">[6]</a> encodes a protein with only 25% aa Id to <i>E</i>. <i>coli</i> MetF. It is more distantly related to the canonical MetF than its chromosomal homolog (Mchl_1881, 56% aa Id to <i>E. coli</i> MetF).</p>d<p>Putative universal stress protein (Mchl_5472) also found in the DCM-dehalogenating <i>M. extorquens</i> DM4 only (METDI4473).</p>e<p>Close homologs (>65% Id aa) located in synteny on the 1.26 Mb megaplasmid of strain AM1.</p